Description: (Taken directly from the application) The apical surface of umbrella cells, the epithelial cells that face the lumen of the urinary bladder, is covered with numerous plaques that have the unique morphology of an asymmetric unit membrane (AUM). At their exoplasmic face they form a crystalline lattice of 16-nm particles that are composed of 4 integral membrane proteins, the uroplakins Ia, Ib, II and III. These crystalline arrays assemble in cytoplasmic vesicles, which may be functionally equivalent to secretory granules but, instead of delivering a secretory protein to the cell surface, they function in the regulated insertion of pre-assembled AUMs into the apical membrane of umbrella cells. Thus, the urothelium, as a machinery devoted to making large amounts of specialized plasma membrane proteins, offers unique opportunities to study the processing, assembly and targeting of such proteins. Our goal is to elucidate the pathways that lead to the assembly of uroplakins into mature AUMs, as well as the mechanisms involved in the delivery of the pre-assembled AUMs to the apical membrane of umbrella cells. We, therefore, will establish the intracellular transport of uroplakins and their oligomeric assemblies using two different cell culture systems, each of which has distinct advantages: Cultured urothelial cells which express all four uroplakins at the cell surface, and 293T cells which do not express uroplakin mRNAs, but they can be very efficiently transfected with one or several of the uroplakin cDNAs. We will investigate the possible phosphorylation of the cytoplasmic domain of UPIII and its possible effect on the assembly of the uroplakin crystals. We also plan to characterize uroplakin domains that are involved in the interaction between uroplakin subunits by applying the yeast two-hybrid system, in which domains of the uroplakins are cloned into the "bait" or "library" vector. In addition, we will perform in vitro translation-translocation experiments, so that we can insert combinations of intact uroplakins or their modified forms into dog pancreas microsomes. Oligomers will be identified by co-immunoprecipitation, by their sedimentation characteristics in sucrose density gradients and by chemical crosslinking approaches. Finally, we will identify urothelium-specific Rab proteins that may be specifically involved in the targeted fusion of AUM vesicles to the apical plasma membrane. These studies should yield new insights into the assembly process of uroplakins and how this process is regulated in normal and hyperpastic urothelium.